Somatotrophic axis plays a key role in the control of metabolic regulation and countless other physiological processes (Renaville et al. Reference Renaville, Hammadi and Portetelle2002). Growth hormone (GH) is not only the major stimulator of postnatal growth, but also affects milk production in cattle (Etherton & Bauman, Reference Etherton and Bauman1998) and regulates the expression of milk protein. At the tissue level, binding of GH to the growth hormone receptor (GHR) activates the Janus kinase 2 (JAK2) pathway. Activated JAK2 in turn induces signal transducer and activator of transcription 5 (STAT5) through multiple phosphorylations and STAT5 is translocated from the cytoplasm to the nucleus, where it stimulates transcription upon binding to specific DNA regions (Herrington & Carter-Su, Reference Herrington and Carter-Su2001). In consequence, expression of insulin-like growth factor I (IGF-I) in the mammary fat pad is mainly regulated by GH (Walden et al. Reference Walden, Ruan, Feldman and Kleinberg1998). In the mammary fat-pad, IGF-I is also required for the terminal differentiation of pre-adipocytes into adipocytes (Blüher et al. Reference Blüher, Kratzsch and Kiess2005). Both IGF-I and IGF type 1 receptor (IGF-1R) have been shown to play a role in the formation and proliferation of terminal end buds, ductal outgrowth and branching during puberty (Sun et al. Reference Sun, Shushanov, LeRoith and Wood2011). It is also known that the IGF1R signal transduction is primarily mediated by the activation of the Ras-Raf-MAP kinase and phosphoinositide 3-kinase (PI3 K)/Akt pathways, wherein IGF-I acts as a strong mitogen inducing cell growth and proliferation, but inhibiting apoptosis (Denley et al. Reference Denley, Cosgrove, Booker, Wallace and Forbes2005). Additional IGF-1R signalling regulates the expression of IRS-1 and IRS-2, which is essential for mammary gland alveolar development (Sun et al. Reference Sun, Shushanov, LeRoith and Wood2011).
Proteins of JAK family members possess seven highly conserved domains referred to as JAK homology domains (JH). The C-terminal domain (JH1) contains a typical tyrosine kinase domain preceded by a pseudokinase domain (JH2), which is considered catalytically inactive. The N-terminal portion of the JAKs (spanning JH7 to JH3) is important for the receptor binding and non-catalytic activity (Silvennoinen et al. Reference Silvennoinen, Ungureanu, Niranjan, Hammaren, Bandaranayake and Hubbard2013). The bovine JAK2 gene is located on BTA8, consists of a total of 25 exons with a long 5′-untranslated region (5′-UTR), spanning over 119 kb and encoding 1132 aa. The translation start codon (AUG) is located in exon 3 (http://www.ncbi.nlm.nih.gov/gene/525246).
There is very little data available in the literature on the potential regulatory role of the JAK2 gene in mammary gland development. Therefore, the objective of this study was to evaluate the effect of the JAK2 gene polymorphism on selected milk production traits in different cattle breeds.
Materials and methods
A total of 904 blood samples were collected from four purebred dairy or dual-purpose cow breeds, including Polish Holstein-Friesian (PHF; N = 224, herd #1A n = 160 (the West Pomeranian province, Poland) and herd #2A n = 64; the Pomerania province, Poland), Montbeliarde (MO; N = 211, herd#1B n = 162 and herd #2B n = 49; the West Pomeranian province, Poland), Simmental (SM; N = 235, herd#1C n = 158 and herd #2C n = 77; the Pomerania province, Poland) and Jersey (JE; N = 234; one herd; Wielkopolska province, Poland). The calvings occurred between 2002 and 2013.
The PHF and MO cows were housed in free-stall barns without access to pasture and fed a total mixed ration (TMR). They were divided into groups according to lactation stage and milk yield. Milking was carried out twice daily in a side-by-side (PHF) or herringbone (MO) milking parlour. The SM and JE cows were housed in two-row stanchion barns and grazed at during the summer period (from May to October). Feeding was based on the partially mixed ration (PMR) system with cows grouped according to their lactation stage and milk yield. Milking was carried out twice daily using the DeLaval milking pipeline machine.
Genomic DNA was isolated from the blood using a MasterPure™ DNA Purification Kit (Epicentre Technologies) according to the manufacturer's instructions.
Primers were designed to PCR-amplify a fragment of the JAK2 gene exon 20 based on a comparison between the whole human gene sequence (GenBank Acc. No. NG_009904) and bovine sequences: Bos taurus Janus kinase 2 transcript variant 2 (GenBank Acc. No. XM_003586385), Bos taurus breed Hereford chromosome 8 and the whole genome shotgun sequence (GenBank Acc. No. DAAA02022688). The rs110298451 SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Table 1).
Table 1. Characterisation of single nucleotide polymorphism under study
* Analogue to rs110298451 Chr8:g.39645396T>C according to Bos_taurus_UMD_3·1 (Note: complementary strand)
PCR reaction mixtures contained the aforementioned genomic DNA as a template, 2·0 μl of PCR buffer with (NH4)2SO4, 2·0 μl dNTP, 1·2 μl MgCl2 (FERMENTAS, ABO Gdansk, Poland), 0·1 μl forward primer (10 pmol/μl) and 0·1 μl reverse primer (10 pmol/μL)(IBB PAN, Warsaw, Poland), 0·5 units of Taq DNA polymerase (FERMENTAS, ABO Gdansk, Poland) and nuclease-free deionised water (Epicentre Technologies, Madison, USA) to a total volume of 20 μl. The following PCR protocol was used: an initial denaturation of 5 min. at 94 °C; 33 cycles of: 94 °C for 50 s, 60 °C for 1 min., 72 °C for 50 s and a final extension at 72 °C for 7 min. After digestion (Table 1), 10 μl of each product was separated by electrophoresis in 2% ethidium bromide-stained agarose gels (Basica Prona™ Agarose, ABO Gdansk, Poland). The gels were visualised under UV and archived.
The data for full 305-d milk production (1st, 2nd, 3rd (PHF and MO) and additional 4th (SM and JE) lactations), including overall milk, fat and protein yields as well as per cent of milk fat and milk protein, were obtained and collected from the official farm records performed with the A4 method (Polish Federation of Cattle Breeders and Dairy Farmers (PFCB&DF), 2014).
Traits of interest were statistically analysed using the general linear model (GLM) procedure. Animals were progeny of up to 79 (PHF), 56 (MO), 111 (SM), 35 (JE) different sires, respectively. For analysis, only first-calf heifers with successive lactations (max to 4 lactations – see: Table 3) were selected. Each year of calving was divided into two seasons namely spring/summer (April to August) and autumn/winter (September to March) which is defined as year/season of calving.
The differences between genotypes were tested using Statistica software (STATISTICA 10·0 PL software package, Statsoft Inc. 2011). The linear model was used as follows:
$$Y_{ijkl} \, = \,{\rm \mu} + G_i + S_j + YS_k + H_l + e_{ijkl} $$where:
Y ijkl – analysed trait; μ – overall mean; G i – fixed effect of JAK2 genotype (i = 1,…3); S j – random effect of a sire; YS k – fixed effect of year-season of calving (the number of classes are included in the Table 3); H l – fixed effect of herd (l = 1, 2 excluded JE); e ijkl – random error.
The effect of breed and number or order of lactation were not included in the model because each of these factors were calculated separately (Table 3).
The χ2 test was used to verify if the allele segregation conformed to the Hardy-Weinberg equilibrium.
Results
In the present study, the occurrence of rs110298451 polymorphism has been validated in 904 individuals. Genotype and allele frequencies of the four breeds are summarised in Table 2. According to the NCBI nucleotide sequence of the JAK2 gene of the Hereford breed (Gene ID: 525246) and other generally available sequences, adenine appears to be the major allele. However, in four dairy breeds, the second allele (JAK2 G) also occurs and all three possible genotypes have been observed. The frequencies of both alleles in the studied herd were usually similar, with the exception of a typical dairy PH-F breed, where homozygous GG genotype was clearly predominant (0·6071), while in two dual-purpose breeds (M and S) animals were generally heterozygous (0·5782 and 0·4936, respectively). Homozygous AA genotype was most frequent (0·4060) in the small-sized milk breed (J), while the remaining two genotypes displayed similar frequencies.
Table 2. The number and frequency of JAK2/RsaI genotypes and alleles in the cows studied
n, number of individuals
The χ2 test showed that the genotype distributions of JAK2/RsaI gene polymorphism were not at Hardy-Weinberg equilibrium (H-WE) in PHF, MO and JE herds. These data indicate a genotypic imbalance in the populations under study where GG genotype frequencies are usually higher than the Hardy-Weinberg expectation at the expense of heterozygous individuals. This could be due to intensive selection, resulting in a tendency towards the accumulation of certain genotypes and the possibility of inbreeding. MO is the exception, probably by the recent introduction and early stage of acclimatisation of both herds.
The analysis of milk performance of all breeds under study divided into particular lactation periods is presented in Table 3. Milk, fat and protein yields in the PHF breed were always the highest in individuals carrying the GG genotype, irrespective of lactation. These results were confirmed statistically. The largest difference was observed between GG and AA genotypes (P ⩽ 0·01). The average differences amounted to approx. +1000 kg of milk, +40 kg of fat and +30 kg of protein. Slightly lower differences were recorded between GG and heterozygous genotypes (P ⩽ 0·01 at 1st and P ⩽ 0·05 in the subsequent lactations).
Table 3. Means and standard errors (se) of milk performance traits in cows with different JAK2/RsaI genotypes for different breeds
Means within rows with the same letters differ significantly at: small letters P ⩽ 0·05; capitals P ⩽ 0·01 Lac – lactation k – the number of classes of year/season n – number of individuals
Similar findings were obtained for Montbeliarde and Simmental breeds. Over the period of three lactations, cows carrying the GG genotype showed the highest yields when compared to the other genotypes. However, statistical differences were found between GG and AA genotypes mainly in milk and protein yields, but sometimes also the fat content. Furthermore, these differences in milk yield were more pronounced in the Montbeliarde breed (+500 ÷ 800 kg) than Simmental cows (+300 ÷ 400 kg during 1st and 2nd lactation and later increased to +1000 kg and even more). Similar results are applicable to fat and protein yields where Montbeliarde cows carrying the GG genotype gave +10 ÷ 15 kg of these components in comparison to cows carrying the AA genotype, whereas Simmental's yields were initially low (+10 kg) and then rapidly increased (about +25 ÷ 50 kg and +25 ÷ 40 kg, respectively). Interestingly, as opposed to Montbeliarde, this elevation of milk yield in the Simmental breed was not reflected in a decrease of fat and protein content (P ⩽ 0·05).
Although unfavourable association of the AA genotype with lower yields has also been detected in the Jersey breed, the differences were limited only to the 1st lactation (e.g., −200 kg of milk compared to the GG genotype).
In brief, irrespective of the breed under study and lactation order, the GG genotype was positively associated with higher milk, protein and fat yields as compared to the AA genotype. Heterozygous individuals were generally characterised by intermediate values of the analysed milk traits, and in certain cases similar or higher to the GG genotype.
Discussion
Among all tyrosine kinase families, certain members of the JAK family play a crucial role in various cellular responses. JAK2 kinase is activated by binding of a ligand to a cytokine receptor (e.g., growth hormone GH-GHR or prolactin PRL-PRLR), triggering phosphorylation of a number of cellular proteins, including associated molecules (cytokine receptors, adaptors, activators or negative regulators) and the JAKs themselves (autophosphorylation) (Argetsinger & Carter-Su, Reference Argetsinger and Carter-Su1996). A total of 49 tyrosine residues are capable of phosphorylation or dephosphorylation (TPA: Janus kinase 2 [Bos taurus] Acc. No. DAA26915), and among them approximately 20 have been intensively investigated. For example, phosphorylation of the activation loop (the Y1007/Y1008 tandem) fully activates the kinase (Feng et al. Reference Feng, Witthuhn, Matsuda, Kohlhuber, Kerr and Ihle1997). The loss of phosphorylation site at Y972 has significant consequences for the whole biological function of JAK2, i.e., phosphorylation of the Y1007/Y1008 tandem (McDoom et al. Reference McDoom, Xianyue, Kirabo, Kuang-Yung, Ostrov and Sayeski2008). A similar activation function is attributed to the tyrosines Y868 and Y966 (all within JH1 domain) (Argetsinger et al. Reference Argetsinger, Stuckey, Robertson, Koleva, Cline, Marto, Myers and Carter-Su2010). During cytokine stimulation, phosphorylation of Y317 (FERM domain) mediates negative-feedback, while on the contrary, Y637 (JH2 domain) is necessary for maximal JAK2 kinase activity (Robertson et al. Reference Robertson, Koleva, Argetsinger, Carter-Su, Marto, Feener and Myers2009). Tyrosines Y221 (FERM domain) and Y570 (JH2 domain) have been shown to modulate JAK2 function in response to GH, wherein the phosphorylation of the former tyrosine increases kinase activity, while the latter reduces it (Argetsinger et al. Reference Argetsinger, Kouadio, Steen, Stensballe, Jensen and Carter-Su2004; Feener et al. Reference Feener, Rosario, Dunn, Stancheva and Myers2004). Kurzer et al. (Reference Kurzer, Argetsinger, Zhou, Kouadio, O'She and Carter-Su2004) showed that phosphorylation of Y813 (JH1 domain) is required for the SH2-Bβ adapter protein to bind JAK2 and to enhance the activity of JAK2 and STAT5B in response to GH. In the context of EpoR signalling, Y119 (FERM domain), Y613 and Y766 seem to play an essential role in binding of JAK2 to this receptor (Funakoshi-Tago et al. Reference Funakoshi-Tago, Pelletier, Matsuda, Parganas and Ihle2006, Reference Funakoshi-Tago, Pelletier, Moritake, Parganas and Ihle2008a). Phosphorylation at Y201 (FERM domain) is necessary for the interaction between JAK2 and SHP-2 (Godeny et al. Reference Godeny, Sayyah, Vonderlinden, Johns, Ostrov, Caldwell-Busby and Sayeski2007). In addition to the above discussed tyrosines, Y570 and serine S523 are constitutively phosphorylated, providing constant negative feedback that blocks kinase activity in the absence of stimulation. However, both amino acids are not conserved in other JAK members, thus the catalytic regulatory function may be unique only to JAK2 (Silvennoinen et al. Reference Silvennoinen, Ungureanu, Niranjan, Hammaren, Bandaranayake and Hubbard2013).
In view of the foregoing observations, localisation of the polymorphism investigated in the present study is not accidental. Based on the results published by other authors regarding the crystal structure of the JAK2 kinase domain, it may be assumed that even small changes in the regions surrounding tyrosines activated in response to GH (i.e., Y813, Y868, Y966, and Y972; Kurzer et al. Reference Kurzer, Argetsinger, Zhou, Kouadio, O'She and Carter-Su2004, Argetsinger et al. Reference Argetsinger, Stuckey, Robertson, Koleva, Cline, Marto, Myers and Carter-Su2010) may be essential for the JAK2 molecule to assume a maximally active conformation. Lysine K912 precedes another important functional phosphorylation site, i.e., tyrosine Y913. According to studies performed by Funakoshi-Tago et al. (Reference Funakoshi-Tago, Tago, Kasahara, Parganas and Ihle2008b), phosphorylation of this tyrosine, located in the JH1 domain, may be involved in the negative regulation mechanism common for JAK family, involving STAT5 activation and signalling. Given the fact that rs110298451 polymorphism does not affect the amino acid sequence, it may only be associated with the causative mutation.
In human, substitution of valine by phenylalanine at codon 617 in the pseudokinase domain (V617F) is the most commonly described polymorphism in JAK2. Although the function of JH2 is not yet well established, this domain is considered a suppressor of catalytic activity of the adjacent tyrosine kinase domain (Babon et al. Reference Babon, Lucet, Murphy, Nicola and Varghese2014). Hyperkinetic kinase activity is known to be the cause of a variety of disorders. For example, in human the V617F mutation causes a number of haematological and myeloproliferative disorders such as polycythaemia vera (PV) or myeloproliferative neoplasms (MPN) (Wolf et al. Reference Wolf, Eulenfeld, Gäbler, Rolvering, Haan, Behrmann, Denecke, Haan and Schaper2013). In Bos taurus, this mutation (exon 14; including UMD 3·1 and Btau_4·6·1) has not been reported thus far. However, two potential SNPs (silent Chr8:g.39652352G>A rs210330018 Cys675 and missense Chr8:g.39652267T>C rs210148032 Ile704Val ATT → GTT) have been submitted within exon 16 that also encodes JH2 pseudokinase. Furthermore, one silent polymorphism rs211067160 has been reported (exon 23, Chr8:g.39634642T>C) in the region encoding kinase domain (JH1; Pro1057). Two additional SNPs were detected in exon 3, i.e., rs377943180 (silent Chr8:g.39719696T>C; Glu12, part of the signal peptide) and rs383317698 (silent Chr8:g39719555A>C; Thr59, part of the JH7 in the FERM domain). Unfortunately, there is no insightful data on the frequency and validation status of those SNPs. The same applies to rs110298451, which was the subject of this research.
We have attempted to capture potential associations between the polymorphism in the bovine JAK2 gene and milk production traits. Despite the fact that the mutation at rs110298451locus is a synonymous substitution we have found non-random correlations in the individuals carrying at least one copy of the JAK2 G allele and their higher productivity in comparison to AA homozygotes. In the following studies we plan to investigate the occurrence of the only known missense mutation (rs210148032) in the same breeds and its possible association with rs110298451.
